Dilute working stocks of libraries and double-stranding primers to 10uM

Dilute working stocks of sequencing primers to 3.2uM (6.4uL of stock solution in 193.6uL water)

Some considerations:

Oligos should be the maximum length because this will help with PCR cleanup and ligation efficiency

Make sure you have some spacer sequence around the restriction site. NEB has a list of the length of the spacer sequence required for each restriction enzyme. (8bp is usually a safe bet)

Order the lowest concentration allowable for the size oligo you want – this will be 50nmole for the 100bp oligo. This will already be more than you’ll need.

If you don’t mind spending more money you can order special “doped” oligo pools where instead of even concentrations of A/T or A/T/C/G or A/T/C, you get 90%A/2%C/8%G, etc. This allows for you to generate a library which is much more likely to produce productive clones.

Double strand the library with modified PCR

Expected max library size is 108 molecules (limit set by transformation efficiency.) You want to load 10X the expected library size for a single library construction. Therefore, you would like to have 109 molecules for a single transformation.

1 pmol corresponds to ~1011 molecules

Use 25pmol of library to make enough for 2500 transformations

Total library DNA should be less than ~25pmol per 100uL reaction

Reaction Mix (100uL, 25pmol library)

Use the following reaction mix for each PCR reaction:

10 μl 10x Thermo polymerase buffer

10 μl 10x dNTPs (10x = 2.5 mM each dNTP)

5 μl 10 μM FWD primer

5 μl 10 μM REV primer

1 μl Polymerase (taq or vent)

66.5 μl H2O

2.5 μl 10μM library stock

PCR protocol

95 C for 2.5 minutes

Cycle 5 times:

55 C (or whatever temperature is appropriate) for 30 s (annealing)

72 C for 1.5 minutes (elongation)

72 C for 10 minutes (final elongation)

4 C forever

PCR cleanup on the double-stranded libraries

This concentrates the samples and allows for the buffer to be switched to something more appropriate.

PCR purification columns can handle up to 10ug of DNA (100pmol of a 100bp oligo is about 3ug)

Expected recovery from a PCR purification reaction is 90% (from the Invitrogen package)

Alternatively, you can run a sample of the first PCR reaction out on a gel for analysis against a sample of the original library (double stranded should run slightly faster than single stranded), then perform the digest. Doing a PCR cleanup on the digest will remove the cut ends, since they are small.